Icosabutate Exerts Beneficial Effects Upon Insulin Sensitivity, Hepatic Inflammation, Lipotoxicity, and Fibrosis in Mice

Icosabutate Exerts Beneficial Effects Upon Insulin Sensitivity, Hepatic Inflammation, Lipotoxicity, and Fibrosis in MiceHepatology CommuniCations, Vol. 0, no. 0, 2019
Icosabutate Exerts Beneficial Effects Upon Insulin Sensitivity, Hepatic Inflammation, Lipotoxicity, and Fibrosis in Mice Anita M. van den Hoek ,1 Elsbet J. Pieterman,1 José W. van der Hoorn,1 Marta Iruarrizaga-Lejarreta,2 Cristina Alonso,2 Lars Verschuren,3 Tore Skjæret,4 Hans M.G. Princen,1 and David A. Fraser4
Icosabutate is a structurally engineered eicosapentaenoic acid derivative under development for nonalcoholic steatohepa- titis (NASH). In this study, we investigated the absorption and distribution properties of icosabutate in relation to liver targeting and used rodents to evaluate the effects of icosabutate on glucose metabolism, insulin resistance, as well as hepatic steatosis, inflammation, lipotoxicity, and fibrosis. The absorption, tissue distribution, and excretion of icosabutate was investigated in rats along with its effects in mouse models of insulin resistance (ob/ob) and metabolic inflamma- tion/NASH (high-fat/cholesterol-fed APOE*3Leiden.CETP mice) and efficacy was compared with synthetic peroxisome proliferator-activated receptor α (PPAR-α) (fenofibrate) and/or PPAR-γ/(α) (pioglitazone and rosiglitazone) agonists. Icosabutate was absorbed almost entirely through the portal vein, resulting in rapid hepatic accumulation. Icosabutate demonstrated potent insulin-sensitizing effects in ob/ob mice, and unlike fenofibrate or pioglitazone, it significantly reduced plasma alanine aminotransferase. In high-fat/cholesterol-fed APOE*3Leiden.CETP mice, icosabutate, but not rosiglitazone, reduced microvesicular steatosis and hepatocellular hypertrophy. Although both rosiglitazone and icosabu- tate reduced hepatic inflammation, only icosabutate elicited antifibrotic effects in association with decreased hepatic con- centrations of multiple lipotoxic lipid species and an oxidative stress marker. Hepatic gene-expression analysis confirmed the changes in lipid metabolism, inflammatory and fibrogenic response, and energy metabolism, and revealed the involved upstream regulators. In conclusion, icosabutate selectively targets the liver through the portal vein and demonstrates broad beneficial effects following insulin sensitivity, hepatic microvesicular steatosis, inflammation, lipotoxicity, oxidative stress, and fibrosis. Icosabutate therefore offers a promising approach to the treatment of both dysregulated glucose/lipid metabolism and inflammatory disorders of the liver, including NASH. (Hepatology Communications 2019;0:1-15).
Although treatment with high-dose oral (4  g/day) eicosapentaenoic acid (EPA) ethyl ester was recently shown to markedly reduce
major adverse cardiovascular events in subjects with elevated triglycerides,(1) there is little evidence of ben- eficial effects of ω-3 fatty acids in other metabolic
Abbreviations: AA, arachidonic acid; ALT, alanine aminotransferase; AUC, area under the curve; DAG, diacylglycerol; EPA, eicosapentaenoic acid; ET-1, endothelin 1; FFA, free fatty acid; GSH, glutathione; GSSG, oxidized glutathione; H&E, hematoxylin and eosin; HETE, hydroxyeicosatetraenoic acid; HOMA-IR, homeostasis model assessment of insulin resistance; IL, interleukin; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PPAR, peroxisome proliferator-activated receptor; STAT1, signal transducer and activator of transcription 1; TNFR, tumor necrosis factor receptor.
Received August 22, 2019; accepted November 11, 2019. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep4.1453/suppinfo. This work was supported by Pronova Biopharma AS/BASF and NorthSea Therapeutics and the TNO research program “Biomedical Health.”
NorthSea Therapeutics was involved in the study design and preparation of the manuscript (D.A.F, T.S), but had no role in the data collection. © 2019 The Authors. Hepatology Communications published by Wiley Periodicals, Inc., on behalf of the American Association for the Study of Liver
Diseases. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modif ications or adaptations are made.
View this article online at wileyonlinelibrary.com. DOI 10.1002/hep4.1453
Potential conflict of interest: Dr. Alonso is employed by OWL Metabolomics. Dr. Fraser is employed by NorthSea Therapeutics, which owns the rights to icosabutate. Dr. Iruarrizaga-Lejarreta is employed by OWL Metabolomics. Dr. Princen received grants from Pronova Biopharma AS/BASF and NorthSea Therapeutics. Dr. Skjaeret is employed by NorthSea Therapeutics, which owns the rights to icosabutate.
[Correction added 6 January 2020 to Table 1, Part A. The heading for Column 3 was incorrect and has been updated.]
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and inflammatory conditions such as diabetes and nonalcoholic steatohepatitis (NASH). For example, in a recent 1-year intervention study, EPA ethyl ester (1.8 or 2.7  g/day) had no effect on liver histology in NASH patients, and clinical trials examining the effects of high-dose (>2 g/day) ω-3 fatty-acid supple- mentation on glycemic control are inconclusive.(2)
A potential explanation for the lack of efficacy of ω-3 fatty acids in conditions beyond cardiovascular disease is that long-chain fatty acids have absorption, distribution, and metabolism properties that prevent optimal liver targeting. First, regardless of the oral delivery form (free fatty acid, ethyl ester, triglycer- ide, or phospholipid), systemic absorption of long- chain fatty acids occurs primarily through lymphatic vessels(3-5) rather than the portal vein, resulting in systemic distribution into adipose tissue, skeletal mus- cle, and many other organs. The consequence of this absorption route is that only 8%-16% of dietary fat reaches the liver after a meal.(6) Second, as high doses of the more potent long-chain fatty acids markedly increase their own oxidation as an energy source,(7) the potential for achieving a dose-dependent effect in the liver is severely limited. Finally, after reaching the liver, extracellular and intracellular receptor bind- ing and eicosanoid pathway metabolism are medi- ated through nonesterified fatty acids (FFAs). Thus, the rapid incorporation of long-chain fatty acids into complex lipids such as triglycerides and phospholipids (in addition to hepatic β-oxidation for energy) may further decrease the availability of the most active free acid form in the liver.
Icosabutate, a structurally engineered EPA derivative in clinical development for NASH (NCT04052516), is designed to overcome the inherent drawbacks of unmodified EPA for liver targeting. It is structured (1) to remain in a free acid form by resisting
incorporation into complex cellular lipids (through an ethyl group in the α-position) and (2) to minimize metabolism through β-oxidation (using an oxygen atom substitution in the β-position), with a goal of achieving therapeutic efficacy beyond what is possible with oral dosing of unmodified ω-3 fatty acids (Fig. 1). Icosabutate was initially developed as a hypolipidemic drug and demonstrated robust reductions in atherogenic lipids and apolipoproteins at a dose of 600  mg once daily in two recent clinical trials.(8,9) Interestingly, icos- abutate also induced a marked and significant reduction in homeostasis model assessment of insulin resistance (HOMA-IR),(8) an effect not typically associated with unmodified ω-3 fatty-acid supplementation.(10) This suggests that icosabutate’s structural modifications not only facilitate quantitative advantages (i.e., low oral dose) but also confer qualitative differences in pharma- codynamic effects versus unmodified EPA.
To further understand icosabutate’s pharmacokinetic and pharmacodynamic profile, we carried out a range of preclinical studies addressing the absorption, dis- tribution, and excretion characteristics of icosabutate. We also performed studies on its effects on glucose metabolism and insulin resistance, as well as steatosis, inflammation, lipotoxicity, oxidative stress, and fibrosis in relevant mouse models. In addition, bioinformatics analysis was used to study the underlying mechanisms involved in the observed hepatic effects.
Methods animals anD eXpeRimental Design
All animal care and experimental procedures were approved by the local Ethical Committee on Animal
aRtiCle inFoRmation: From the 1 Department of Metabolic Health Research,  The Netherlands Organization for Applied Scientific Research, Leiden, the Netherlands; 2 OWL Metabolomics,  Parque Tecnológico de Bizkaia, Derio, Spain; 3 Department of Microbiology and Systems Biology,  The Netherlands Organization for Applied Scientific Research, Zeist, the Netherlands; 4 NorthSea Therapeutics BV, Naarden, the Netherlands.
aDDRess CoRResponDenCe anD RepRint ReQuests to: Anita M. van den Hoek, Ph.D. TNO Metabolic Health Research Zernikedreef 9
2333 CK Leiden, the Netherlands E-mail: [email protected] Tel.: +31-888-666-021
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Care and Experimentation and were in compliance with European Community specifications regard- ing the use of laboratory animals. All animals were group-housed in a temperature-controlled room on a 12-hour light-dark cycle and had free access to food and water. Body weight, food intake (both between 8 and 9 am), and blood/plasma measures (after 4 hours of fasting) were regularly monitored during the stud- ies. Animals were sacrificed by CO2 inhalation.
To analyze portal vein uptake of icosabutate, 8-week old male Wistar rats (n = 4) were used and were pre- treated with buprenorphine (0.01-0.05  mg/kg, sub- cutaneously) and then anesthetized by pentobarbital (50  mg/kg, intraperitoneally). Rats received a single oral gavage of 100 mg/kg icosabutate, and blood was collected from portal vein and from mesenteric lymph duct over 24 hours.
To analyze tissue distribution and excretion of icos- abutate, male albino Wistar rats (n  =  7 for distribu- tion, n = 3 for excretion) received a single oral gavage of 100 mg/kg [14-C]-icosabutate. Plasma concentra- tions and tissue distribution were measured by liquid scintillation counting and quantitative whole-body autoradiography, respectively, at multiple time points (1, 2, 4, 8, and 24 hours, and 3 and 7 days). Routes and rates of excretion were measured by collection of urine and feces at corresponding time points.
To analyze the effects of icosabutate on obesity, hyperglycemia and insulin resistance, 6-8-week- old male ob/ob mice were randomized into different groups (n  =  10/group) and were left untreated (con- trol group) or treated for 5 weeks with 135 mg/kg/d icosabutate through diet administration. As reference,
groups mice treated for 5 weeks with 100  mg/kg/d fenofibrate or 30  mg/kg/d pioglitazone through diet administration were included. An oral glucose (2 g/kg) tolerance test was performed after 5 weeks, after 4-hour fasting. First, baseline blood samples (t  =  0) were collected from the tail vein into ethylene diamine tetraacetic acid (EDTA)-coated tubes (Sarstedt, Nümbrecht, Germany). Subsequently, mice received a bolus (2  g/kg) of 10% (wt/vol) D-glucose solution through gavage, and additional blood samples (30 µL) were drawn at 15, 30, 45, 60, and 120  minutes after injection.
To analyze the effects of icosabutate on hepatic ste- atosis, inflammation and fibrosis, 8-15-week-old male APOE*3Leiden.CETP mice fed a high-fat (24% wt/wt lard) and high cholesterol (1% wt/wt) diet(14) were randomized into different groups (n = 12/group) and received daily vehicle (corn oil) gavages (control group) or were treated for 20 weeks with 112 mg/kg/d icosabutate through daily oral gavages. As a reference group, mice treated for 20 weeks with 13  mg/kg/d rosiglitazone through diet administration (receiv- ing daily vehicle gavages) were included. NASH and fibrosis were scored in hematoxylin and eosin (H&E) or sirius red–stained cross sections using an adapted grading system of human NASH(11,12) and were ana- lyzed by biochemical analysis, as described in more detail in the Supporting information.
At several time points during the studies, animals were fasted for 4 hours and blood was collected from the tail vein into EDTA-coated tubes (Sarstedt) for different analytical measurements, as described in more detail in the Supporting information.
Fig. 1. Chemical structure of icosabutate as compared with EPA. Icosabutate, a structurally engineered EPA derivative, is structured (1) to remain in a free acid form by resisting incorporation into complex cellular lipids (through an ethyl group in the α-position) and (2) to minimize metabolism by way of β-oxidation (through an oxygen atom substitution in the β position).
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Liver samples were used for histology, hepatic lipid/ lipidomics analysis, assessment of hepatic reduced glutathione (GSH), and oxidized glutathione (GSSG) and transcriptome analysis, all described in more detail in the Supporting information.
statistiCal analysis All values shown represent the means  ±  SEMs.
Statistical differences between groups were deter- mined by using nonparametric Kruskal-Wallis fol- lowed by Mann-Whitney U test for independent samples using SPSS software (IBM Corp., Armonk, NY). A P value < 0.05 was considered statistically significant.
Results iCosaButate eFFeCtiVely taRgets tHe liVeR
To evaluate whether structural alterations of icos- abutate enhanced uptake through the portal vein, absorption after single oral administration was ana- lyzed in male Wistar rats with concurrent collection
of blood from portal vein and lymph from mesenteric lymph duct. Area under the curve (AUC)0-24h and maximal concentration (Cmax) values of 14C-labeled icosabutate were approximately 2-fold higher in the portal vein versus the mesenteric lymph (Table 1A). Accounting for the much higher flow rate of portal vein plasma (522  mL/h) versus mesenteric lymph (0.5  mL/h), the data demonstrate that icosabutate is almost entirely taken up through the portal vein (>99%) with only a small fraction of icosabutate being absorbed through the lymphatic pathway. This con- trasts with the high uptake of unmodified long-chain fatty acids by way of mesenteric lymph after incorpo- ration into chylomicrons.(3-5)
Tissue distribution analysis showed peak concentra- tions of radioactivity in most tissues at 4-8 hours after the dose (except the gastrointestinal tract) with high- est concentrations in the liver and kidney (Table 1B). Most other tissues contained levels of radioactivity below that in plasma, indicating a limited distribution of absorbed radioactivity. There was a rapid decline in concentrations of drug-related material over the study period, with excretion of radioactivity greater than 95% complete by 48 hours, and with 60% and 40% excreted through urine and feces, respectively (data not shown).
taBle 1. poRtal Vein anD mesenteRiC lympH aBsoRption anD tissue DistRiBution oF iCosaButate
A
Mesenteric Lymph (Lymph Flow Rate: 0.5 mL/h)
Portal Vein/Lymph RatioCmax (µg/mL) Tmax (h) AUC0-24h (µgh/mL) Cmax (µg/mL) Tmax (h) AUC0-24h (µgh/mL)
Icosabutate in corn oil
B
Activity (µg/g tissue)
1 Hour 2 Hours 4 Hours 8 Hours 1 Day 3 Days
Liver 77.7 134.0 174.0 145.0 25.5 2.7
Kidney cortex 34.0 70.6 92.8 38.5 9.8 1.5
Kidney medulla 47.3 148.0 102.0 76.6 34.5 0.5
Muscle 3.6 5.2 10 4.8 1.2 BLQ
Subcutaneous fat 9.0 14.6 25.3 15.4 7.1 4.0
Blood 24.6 36.2 60.9 28.1 2.6 BLQ
*Assuming a volume ratio of plasma:blood of 1:2. Abbreviations: BLQ, below the limit of quantification; h, hours.
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iCosaButate impRoVes gluCose metaBolism anD insulin ResistanCe
The effects of 4-week treatment with icosabutate on glucose metabolism and insulin resistance were analyzed in a diabetic mouse model, ob/ob mice. We included fenofibrate to control for potential peroxisome proliferator-activated receptor α (PPAR-α)–mediated effects and added pioglitazone, a PPAR-γ agonist (and to a lesser extent PPAR-α activity(13)), as a positive control. As shown in Fig. 2, icosabutate significantly improved glucose metabolism. This was reflected by a significant decrease in blood glucose, blood hemo- globin A1c, plasma insulin, and HOMA-IR (−50%, −47%, −76% and −87%, respectively; all p  <  0.001). Fenofibrate and pioglitazone also improved glucose metabolism, although effects following plasma insu- lin were less pronounced with fenofibrate. Icosabutate markedly improved glucose tolerance after an oral glucose load (Fig. 2G) and significantly (p  <  0.001) reduced AUC (0-120 minutes) by 60% (Fig. 2H). Fenofibrate had a less potent effect on glucose toler- ance and AUC, whereas pioglitazone reduced AUC to a similar degree as icosabutate. Pioglitazone demon- strated the largest reduction in HOMA-IR (−97%, p  <  0.001) and was the only compound to increase adiponectin (4.5-fold, p < 0.001). In contrast to piogl- itazone, neither fenofibrate nor icosabutate affected body weight (54.4  ±  1.5, 55.0  ±  1.1, 56.6  ±  1.4, and 62.3  ±  1.6 for control, icosabutate, fenofibrate and pioglitazone group, respectively, after 4 weeks of treat- ment) or adiponectin (Fig. 2E). Plasma alanine ami- notransferase (ALT) levels were significantly decreased as compared with the control group by icosabutate treatment only (−33%, p < 0.01) (Fig. 2F).
iCosaButate impRoVes miCRoVesiCulaR steatosis, HepatiC inFlammation, anD FiBRosis
To assess the effects of icosabutate on NASH in a rodent model characterized by more severe hepatic inflammation and mild fibrosis, APOE*3Leiden. CETP mice(14) were fed a high-fat/cholesterol diet and treated with either icosabutate (112 mg/kg/d) or rosiglitazone (13 mg/kg/d) for 20 weeks. Icosabutate prevented microvesicular steatosis (−35%, p  <  0.05)
and hepatocellular hypertrophy (−82%, p < 0.01), but not macrovesicular steatosis (Fig. 3A-D), whereas rosiglitazone did not significantly affect either param- eter. Biochemical analysis of intrahepatic liver lipids (Fig. 3E-G) revealed a significant reduction of hepatic cholesterol ester with icosabutate and rosiglitazone. Both icosabutate and rosiglitazone significantly pre- vented hepatic inflammation, as shown by the reduced number of inflammatory cell aggregates (Fig. 3H), by −62% and −67%, respectively (both p < 0.01). The anti-inflammatory effect of icosabutate was confirmed by measurement of plasma inflammation markers. Plasma monocyte chemoattractant protein 1 was reduced by −30% and −46% for icosabutate and rosigl- itazone (p < 0.01), respectively, although the reduction achieved by icosabutate was not significant (p = 0.123) (111.5 ± 17.1, 78.6 ± 6.4, and 65.1 ± 5.1 for control, icosabutate, and rosiglitazone-treated group, respec- tively, after 20 weeks of treatment). For the liver- derived biomarker serum amyloid A, icosabutate showed a more potent reduction (−68%, p  <  0.001) versus control than rosiglitazone (−46%, p  <  0.01) (29.8 ± 5.0, 9.7 ± 0.6, and 16.1 ± 3.3 for control, icos- abutate, and rosiglitazone-treated group, respectively, after 20 weeks of treatment). Despite comparable decreases in hepatic inflammatory cell aggregates, only icosabutate reduced hepatic collagen content (−32%, p  <  0.01 versus control; Fig. 3J) and fibrosis surface area (−26%, p < 0.05; Fig. 3AI). Representative pictures of fibrosis (sirius red staining) and hepatic lipids (H&E stain) from individual mice from con- trol, icosabutate, and rosiglitazone groups are shown in Fig. 3A. Rosiglitazone significantly increased food intake at multiple time points (p  <  0.05) during the study but did not significantly increase body weight, whereas there was a small but significant decrease in body weight at week 16 and 20 versus control (−15%, p > 0.05) in the icosabutate group despite no change in food intake (data not shown).
iCosaButate, But not RosiglitaZone, ReDuCes HepatiC ConCentRations oF multiple lipotoXiC lipiD speCies anD oXiDatiVe stRess maRKeRs
As icosabutate, but not rosiglitazone, reduced hepatic fibrosis despite comparable effects on influx of
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inflammatory cells, hepatic concentrations of NASH- associated lipotoxic lipid species(15) and oxidative stress were measured in liver tissue of APOE*3Leiden.
CETP mice fed a high-fat/cholesterol diet and treated for 20 weeks. As shown in Fig. 4, icosabutate signifi- cantly reduced hepatic concentrations of multiple
Fig. 2. Icosabutate improves glucose metabolism and insulin sensitivity. Ob/ob mice were left untreated (control) or treated with icosabutate, fenofibrate, or pioglitazone for 5 weeks. Blood glucose (A), plasma insulin (B), blood hemoglobin A1c (C), HOMA-IR (D), and plasma adiponectin (E) and plasma ALT (F) levels were measured after 4 weeks of treatment. Oral glucose tolerance test was performed in 4-hour-fasted ob/ob mice after 5 weeks of treatment. Blood glucose levels (G) were measured before (basal) and 15, 30, 45, 60, and 120 minutes after oral glucose load, and the AUC (H) was calculated. Values represent the mean ± SEM for 10 mice per group (*p < 0.05, **p < 0.01, ***p < 0.001 versus control). Abbreviations: HbA1c, hemoglobin A1c; OGTT, oral glucose tolerance test.
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Fig. 3. Icosabutate improves NASH and liver fibrosis. Histological photomicrographs of liver cross sections stained with H&E or sirius red (A) and quantitative analysis (B-J) of NASH and liver fibrosis in APOE*3Leiden.CETP mice fed a high-fat/cholesterol diet and left untreated (control) or treated with icosabutate or rosiglitazone for 20 weeks. Macrovesicular steatosis (B), microvesicular steatosis (C) and hepatocellular hypertrophy (D) as percentage of total liver area, intrahepatic triglycerides (E), free cholesterol (F) and cholesterol esters (G), inflammatory foci per millimeters-squared microscopic field (H), and fibrosis as percentage of total liver area (I) or as hepatic collagen content ( J) were analyzed. Values represent mean ± SEM for 12 mice per group (*p < 0.05, **p < 0.01, ***p < 0.001 versus control).
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Fig. 4. Icosabutate reduces the hepatic concentrations of lipotoxic lipid species and oxidative stress markers. Icosabutate reduces hepatic concentrations of lipotoxic lipid species: FFAs (A), DAGs (B), bile acids (C), arachidonic acid (D), ceramides (E), and HETEs (F) (comprising 11[R]-, 12-, and 15[S] isomers) in APOE*3Leiden.CETP mice fed a high-fat/cholesterol diet and left untreated (control) or treated with icosabutate or rosiglitazone for 20 weeks. Icosabutate significantly improves the GSH/GSSG ratio (H) through a reduction in hepatic GSSG concentrations (G). No effect on any parameter was noted for rosiglitazone except a significant increase in HETEs. Data are presented as mean ± SEM for 12 mice per group (*p < 0.05, **p < 0.01, ***p < 0.001 versus control).
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lipotoxic lipid…